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S [16]. To reduce complexity, samples can be fractionated or enriched in particular compartments prior to MS analysis [17]. MS may be combined with activity-based probes to determine not only the occurrence and quantification of proteins but also their functional activity [18]. Affinity-based proteomics involving capture agents, notably antibodies, can be utilized to facilitate highthroughput analysis of proteins or for confirmatory studies, for example, using tissue microarrays (Figure 3b) [19]. Antibodies may be spotted onto an array to allow capture and identification of their targets. Alternatively, with reverse-phase protein arrays, lysate samples are spotted onto arrays that are incubated with an antibody against a known target [20]. Proteins and peptides may also be spotted onto microarrays for their interrogation. The depth of analysis currently achievable through proteomics is such that, given a sufficient number of cells, a protein product of virtually any expressed gene in a cell population should be identifiable. The challenge for proteomic studies of the tumor microenvironment stems from the cellular complexity of the microenvironment, necessitating separate analysis of individual cell types. Moreover, the insoluble nature of the matrix complicates analysis of intact proteins, a challenge that can be overcome by first digesting the matrix before MS of the resulting peptide mixture. MS tools are increasingly being utilized to characterize the numerous posttranslational protein modifications that play a role in the tumor microenvironment - for example, through their impact on the cross-linking of matrix proteins [21]. Above all, for studies of the tumor microenvironment to be most informative, they must utilize the in-depth, quantitative proteomic methodologies that are currentlyHanash and Schliekelman Genome Medicine 2014, 6:12 http://genomemedicine.com/content/6/2/Page 3 ofCultured cells Cell surface Biotinylation of surface proteins Secretome +/- exosomes Cell culture media Concentration Total extract Lysis and affinity captureProteinGlycopeptide captureElutionCell lysisPhosphoproteins and other PTMsNuclear and other organelle proteinsFigure 2 Analysis of the proteome of cell populations by sub-compartment. Proteomics is particularly informative when applied to individual cellular compartments, such as the cell surface, secretome, the nucleus or other organelles, for which isolation and analysis procedures are required. Moreover, aside from the identification of constituent proteins, there is a need to Alectinib assess post-translational modifications (PTMs) of proteins, including major modifications such as phosphorylation and glycosylation, in addition to cleavage and proteolysis. Cell surface proteins (for example, receptors and antigens) may be captured through the use of lipid impermeable biotin followed by capture of surface proteins using monomeric avidin and subsequent mass spectrometry (MS) analysis. Proteins in cell culture media may be fractionated as intact proteins using chromatography followed by digestion of individual fractions and MS analysis. Alternatively, particulate material (for example, exosomes) may be first isolated from media, followed by their MS analysis separately from the soluble fraction in the media.available but largely restricted to specialized laboratories and centers.Insights from proteomic profiling of the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/7500280 tumor extracellular matrix The ECM regulates intercellular communication and serves as a re.